Experimental site, plant materials and applied treatments
The experiment was conducted in the research greenhouse of the Faculty of Agriculture, University of Maragheh, Maragheh, Iran (longitude 46°16' E, latitude 37°23' N, altitude 1485 m) as factorial experiment using a completely randomized design (CRD) in three replications. Two-year-old cuttings of grapevine cv. Sultana were planted in 7-kilogram pots containing a mixture of coco peat and medium grain perlite in a ratio of 3:1 (each pot contained a cutting). Then, they were irrigated with ½-strength Hoagland solution until at least eight true leaves emerged. At that point, plants were treated with the chemical priming treatments four times at 12 h intervals. The treatments included putrescine (Put) at two concentrations (5 and 10 mg L-1), carbon quantum dot (CQD) NPs at two concentrations (5 and 10 mg L-1) and putrescine-functionalized carbon quantum dots (Put-CQD NPs) at two concentrations (5 and 10 mg L-1), each treatment in three replications. Treatments were done in combination with Hoagland solution into the culture medium of pots. The last application of priming treatments was performed 48 h prior to imposition of salt stress. Consequently, salinity stress at two concentrations (0 and 100 mM NaCl) was imposed daily through watering with Hoagland solution and continued up to a month. All biochemical and enzymatic measurements were implemented three days after imposition of salt stress using fully expanded leaves. Sampled leaves were instantaneously kept into liquid nitrogen for 2 min and afterwards preserved at -80 °C freezer until measurements were carried out. Leaf samples were used to assay hydrogen peroxide (H2O2), malondialdehyde (MDA), electrolyte leakage (EL), total phenolic compounds, proline, and antioxidant enzymatic activities. Other parameters including Na+/K+ content, photosynthetic parameters and pigments were investigated a month after salinity application. Pigments were examined via the same above-mentioned sampling protocol, while leaf fresh and dry weights and photosynthetic parameters were assayed using fresh leaves. Three technical replications were used for each measurement. Control plants were irrigated simply with ½-strength Hoagland solution.
Preparation of putrescine functionalized carbon quantum dots (Put-CQD NPs)
In a 25 mL Teflon-lined autoclave chamber containing 10 mL distilled water, 0.5 g putrescine and 2 g citric acid were added and heated at 200 °C for 12 h. After cooling the reaction temperature to room temperature, the pH value of resulted red-brown solution was set to 7 by NaOH before use and characterization. For comparison, the same procedure was used to synthesize bare CQDs.
Leaf fresh and dry weights
Five leaf samples were individually weighed for fresh weight (FW) and then kept in the oven (70 °C, 72 h) for dry weight (DW) measurements at the harvest stage.
Na+ and K+ assay
Leaf samples were randomly collected from each treatment, washed and air dried and then dried in hot-air oven at 60°C for 18 h. Afterwards, the samples were ground in Willey mill and the powered samples were stored for the assay. The triacid digestion extract was used for estimation of Na+ and K+ by flame photometry as out-lined by Ghosh  and expressed in mmol kg–1. Thereby, Na+/K+ ratio was determined.
Quantification of photosynthetic pigments (chlorophyll a, b and carotenoids)
Fully expanded leaves (0.2 g) were extracted in 0.5 mL acetone (3% v/v) and then centrifuged (10000 rpm, 10 min) and the absorption of the obtained supernatant was recorded at 645 nm (Chl b), 663 nm (Chl a) and 470 nm (carotenoids) by UV-Vis spectrophotometry (UV-1800 Shimadzu, Japan). Chl a, b and carotenoids contents were calculated through the equations described by Sharma et al. .
Chlorophyll fluorescence and SPAD assay
A dual-pam-100 chlorophyll fluorometer (Heinz Walz, Effeltrich, Germany) was used to measure chlorophyll fluorescence parameters including Fv/Fo, Fv/Fm and Y (II). The measurement was done after the plants were dark-adapted for 20 min .
Five randomly selected leaves of each pot were used to determine SPAD values (leaf chlorophyll concentrations) via a SPAD-meter (502 Plus Chlorophyll Meter, Japan) .
Electrolyte leakage (EL) assay
For EL assay, 0.5-cm diameter discs of fully expanded leaves were cut; the discs were then washed thrice by deionized water and incubated in ambient temperate for 24 h. A conductivity meter (Hanna, HI98192) was used to measure the initial electrical conductivity (EC1) of the solution. At that time, the samples were incubated in a water bath (95 °C, 20 min) to release all electrolytes, cooled down to 25 °C and their final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was calculated from following equation .
EL (%) = (EC1/EC2) × 100
Malondialdehyde (MDA) and hydrogen peroxide (H2O2) assay
After homogenizing 0.1 g leaf samples with 2.5 mL acetic acid (10% w/v) and centrifuging (15,000 rpm, 20 min), the same volume of the obtained supernatant and thiobarbituric acid (0.5% w/v) in trichloroacetic acid (TCA) (20%) was incubated at 96 °C for 30 min in the test tube. Samples were then placed at 0 °C for 5 min and centrifuged (10,000 rpm, 5 min) and the absorbance was recorded at 532 and 600 nm by the spectrophotometer. MDA content was calculated using the following equation:
MDA (nmol g-1 FW) = [(A532-A600) ×V×1000/ɛ] ×W
Note: ɛ= the specific extinction coefficient (155 mM-1cm-1), V= the volume of crushing medium, W= the leaf FW, A 600= absorbance at 600 nm and A 532= the absorbance at 532 nm .
To assay H2O2, 0.2 g leaves were finely mixied with 5 mL trichloroacetic acid (0.1 % w/v) in an ice bath and then centrifuged (12000 rpm, 4oC, 15 min). To the obtained supernatant (0.5 ml), 0.5 mL potassium phosphate buffer (pH 6.8, 10 mM) and 1 mL potassium iodide (1 M) were added and the absorbance was recorded at 390 nm. Finally, H2O2 content was calculated by standard calibration curve previously made by various H2O2 concentrations and expressed as µmol g-1 FW .
To assay proline content, 0.5 g leaf samples were homogenized in 10 mL aqueous sulfosalicylic acid (3%) in an ice bath. After centrifuging (1000 rpm, 4°C), 2 mL ninhydrin acid and 2 mL g lacial acetic acid (a 1:1:1 solution) were added to 2 mL supernatant, finely mixed and incubated at 100 ºC for 1 h. The reaction was stopped in an ice bath and finally 4 mL toluene was added and mixed vigorously (20 s). The mixture absorbance was recorded at 520 nm using the spectrophotometer. Different concentration of L-proline was used for standard curve and final calculation of proline values .
Quantification of total phenolic compounds
Brieﬂy, after digesting 0.1 g leaf sample with 5 mL 95% ethanol, the mixture was kept in dark (24 h) and then to 1 mL of supernatant, 1 mL 95% ethanol and 3 mL distilled water were added. Next step was adding 0.5 mL 50% Folin-Ciocalteu solution and 1 mL 5% sodium bicarbonate, and after 1 h in the dark, the absorbance was recorded at 725 nm using the spectrophotometer. The absorbance values were converted to total phenols through standard curve made by different concentrations of gallic acid and expressed as mg gallic acid (GAE) g-1 FW .
Assay of antioxidant enzymatic activities
Total soluble proteins and antioxidant enzymes activities were assayed through leaves formerly stored at -80 °C freezer. All steps of enzyme extraction were carried out at 4 °C as follows: leaves (0.5 g) were homogenized with potassium phosphate buffer (pH 6.8, 100 mM) containing 1 % polyvinylpyrrolidone (PVP) and EDTA (4 mM) using magnetic stirrer for 10 min. After centrifuging (6000 rpm, 20 min), the supernatant was collected to evaluate total soluble proteins, catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and guaiacol peroxidase (GP) enzymatic activities based on the same procedures described by Gohari et al. (2020b).
All obtained data analysis was performed by SAS software and the means of each treatment were analyzed by Duncan’s multiple range test at the 95% level of probability (SAS Institute Inc., ver. 9.1, Cary, NC, USA).